PROJECT/SUMMARY ABSTRACT Early-stage triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype in which, despite standard of care, approximately 1 in 3 patients relapse within the first three years of diagnosis. Through our group?s previous identification of the glucocorticoid receptor (GR) signaling pathway as a risk factor for recurrence in TNBC, we discovered that the gene encoding nicotinamide N-methyltransferase (NNMT) is a direct GR transcriptional target. We also found that high NNMT expression, independently of GR expression, is associated with a significantly worse outcome in early-stage chemotherapy-treated TNBC, suggesting a role for high NNMT activity in tumor cell chemotherapy-resistance and metastasis. Recently, accumulating evidence suggests that NNMT activity may play a role in the biology of various human cancers; however, NNMT?s mechanism of action in TNBC is completely unknown. Based on our preliminary data suggesting that NNMT activity induces hypomethylation of an important subset of TNBC mRNAs, we hypothesize that NNMT alters an oncogenic epitranscriptome of aggressive TNBC. Our central hypothesis is that high NNMT activity promotes TNBC cell survival and metastasis by reducing m6A mRNA modification of target transcripts, increasing oncogenic mRNA stability, and upregulating resultant cell survival and metastatic protein expression. In Aim 1, we will determine whether NNMT depletion or inhibiting NNMT activity biochemically alters m6A mRNA modification. We will also determine whether high versus depleted NNMT-expressing TNBC cells exhibit differential motility/invasion, adherence-independent viability and protection from chemotherapy- induced apoptosis in two dimensional (in vitro) assays. In Aim 2, using control, NNMT-knockdown, and NNMT- overexpression-rescue TNBC xenografted cells and a syngeneic model, we will determine whether NNMT expression/activity contributes to altered expression of m6A mRNA modified tumor-promoting genes and increases tumor regrowth. The same experiments will also be performed with and without NNMT inhibitor treatment. In summary, NNMT is a putative driver of epitranscriptomic mRNA modification that we will test as a driver of chemoresistance and metastatic gene expression and will test as a novel therapeutic target in TNBC.